(Hypertension. 1997;30:1112-1120.)
© 1997 American Heart Association, Inc.
Articles |
Arginine Vasopressin Increases Nitric Oxide Synthesis in Cytokine-Stimulated Rat Cardiac Myocytes
From the Departments of Cardiology (K.Y., U.I., K.S.) and Endocrinology and Metabolism (K.O., T.S.), Jichi Medical School, Minamikawachi, Tochigi, Japan, and the Department of Internal Medicine (Y.K., M.O., M.Y.), First Division, Kobe University School of Medicine, Hyogo, Japan.
Correspondence to Uichi Ikeda, MD, Department of Cardiology, Jichi Medical School, Minamikawachi, Tochigi 329-04, Japan. E-mail uikeda{at}jichi.ac.jp
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Abstract We investigated the effects of arginine vasopressin (AVP) on nitric oxide (NO) synthase activity in cardiac myocytes by measuring the production of nitrite, a stable metabolite of NO, and the expression of inducible NO synthase (iNOS) mRNA and protein. Incubation of cultured neonatal rat cardiac myocytes for 24 hours with interleukin-1ß (IL-1ß) caused a significant increase in NO production. Both AVP and V1a receptor agonist [Phe2,Ile3,Orn8]vasopressin augmented NO synthesis in IL-1ß–stimulated, but not in unstimulated myocytes, in a dose-dependent manner. The V1a receptor antagonist [d(CH2)51,O-Me-Tyr2,Arg8]vasopressin completely inhibited the effect of AVP. The AVP-induced NO production by IL-1ß–stimulated cells was accompanied by increased iNOS mRNA and protein accumulation. AVP caused a significant increase in cytosolic free Ca2+ levels of cardiac myocytes, whereas it showed no effect on cytosolic cAMP levels. After protein kinase C activity was functionally depleted by treating cells with phorbol 12-myristate 13-acetate for 24 hours, AVP did not augment IL-1ß–induced NO production. The effect of AVP was also inhibited in the presence of the protein kinase C inhibitor calphostin C. The addition of AVP increased protein kinase C activity in cardiac myocytes, and its effect was significantly inhibited in the presence of calphostin C. These results support the hypothesis that the heart may be a target organ for AVP and that AVP modulates IL-1ß–induced iNOS expression in myocytes through the V1a receptor, which is mediated at least partially via activation of protein kinase C.
Key Words: interleukins • endothelium-derived factors • calcium
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Introduction |
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Nitric oxide, short-lived radical gas, plays an important role as an intercellular messenger in most or all mammalian organs, participating in vascular homeostasis, neurotransmission, and antimicrobial defense. Three genes encode NOS of two biochemical types. Type I nNOS and type III eNOS are constitutive but dormant until activated briefly by Ca2+ transients that sustain the binding of calmodulin. A third NOS is expressed only after transcriptional induction.1 Once synthesized, this NOS is active for prolonged periods, perhaps because it binds calmodulin without a requirement for elevation of Ca2+ above the levels in resting cells.2 Termed iNOS, or type II NOS, the latter form of the enzyme, has been purified3 4 and cloned5 6 from mouse macrophages. iNOS is upregulated in several tissues by lipopolysaccharide and cytokine stimulation, where it generates the sustained release of large amounts of NO.7 Several previous studies have demonstrated that NO can modulate myocardial contractility.8 9 10 11 12 13 Hosenpud et al8 reported that IL-1ß exhibited a negative inotropic effect on canine hearts in vivo through NO production. Roberts et al9 and we13 have reported that IL-1ß showed a suppressive effect on beating rate of cultured rat cardiac myocytes via NO production. In humans, cytokine induction of myocardial iNOS with reduced cardiac contractility may also be present in sepsis and after the use of cytokines as antitumor therapy.14 15 Recently, Haywood et al16 and Habib et al17 reported that iNOS gene expression occurred frequently in failing human cardiac myocytes of patients with dilated cardiomyopathy, ischemic heart disease, and valvular heart disease.
The antidiuretic hormone AVP is a cyclic nonapeptide involved in the cardiovascular homeostasis of body fluid osmolarity, blood volume, vascular tone, and blood pressure. AVP also belongs to the family of vasoactive and mitogenic peptides involved in physiological and pathological cell growth and differentiation. AVP exerts its actions through binding to specific V1a, V1b, and V2 membrane receptors coupled to distinct second messengers. The V1a receptor, which mediates cell contraction and proliferation, platelet aggregation, and glycogenolysis, is localized in vascular smooth muscle cells, hepatocytes, platelets, and mesangial cells.18 19 20 Recently, cDNAs encoding rat and human V1a receptors have been cloned.21 22 The V1b receptor is located in the anterior pituitary where it stimulates corticotropin release.23 cDNAs encoding rat and human V1b receptors have also been cloned.24 25 The V1 receptors activate phospholipases A2, C, and D, resulting in the production of inositol 1,4,5-triphosphate and 1,2-diacylglycerol, mobilization of intracellular Ca2+, influx of extracellular Ca2+, activation of protein kinase C, and protein phosphorylation.26 On the other hand, the V2 receptor is localized on the renal tubular epithelial cells and mediates increases in the formation of cAMP.27 This receptor is responsible for the antidiuretic effect that is normally used as an indicator of the V2 activity. Recently, Lolait et al28 and Birnbaumer et al29 described the molecular cloning of rat and human V2 receptors.
There are few studies30 31 32 that have investigated the interactions between NO and AVP. In patients with congestive heart failure, plasma AVP levels are frequently increased,33 34 and a negative correlation has been reported between ejection fraction of the left ventricle and plasma AVP levels.35 However, the precise role of the elevated AVP, whether the heart is a target organ of AVP, and the interaction between NO and AVP in the heart remain unknown. In the present study, we investigated the effects of AVP on NO synthesis in cultured neonatal rat cardiac myocytes.
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Methods |
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Reagents
[Arg8]vasopressin (AVP) was purchased from Peptide Institute. The selective V1a receptor agonist [Phe2, Ile3,Orn8]vasopressin was purchased from Peninsula Laboratories. The selective V1a receptor antagonist [d(CH2)51,O-Me-Tyr2,Arg8]vasopressin, the selective V2 receptor agonist 1-deamino-8-D-AVP and calphostin C were purchased from Kyowa Hakko Kogyo. The selective V1b receptor agonist [deamino1,D-3-(pyridyl)Ala2, Arg8]vasopressin was purchased from Bachem. A polyclonal anti-iNOS antibody, which was raised against rat liver iNOS,36 was kindly provided by Dr H. Esumi (National Cancer Center Research Institute, Tokyo, Japan). Donkey anti-rabbit IgG antibody conjugated with horseradish peroxidase was purchased from Amersham International Plc. Human recombinant IL-1ß was a gift from Otsuka Pharmaceutical Co (Tokushima, Japan). L-NMMA, actinomycin D, lipopolysaccharide (LPS; 026:B6), angiotensin II, PMA, and IBMX were purchased from Sigma Chemical Co. The acetoxymethyl ester of fura-2 (fura-2/AM) was obtained from Dojin Biochemicals. Other materials and chemicals were obtained from commercial sources.
Cell Culture
Cardiac myocytes were prepared from ventricles of 1-day-old
Sprague-Dawley rats as described previously.37 Briefly,
after dissociation with 0.25% trypsin, cell suspensions were washed
with DMEM (GIBCO Laboratories) supplemented with 10% FBS, and
centrifuged at 500g for 10 minutes. The
centrifuged cells were then resuspended in 10% FBS containing
DMEM. For selective enrichment of cardiac myocytes, the dissociated
cells were preplated for 1 hour, during which time nonmyocytes
readily attached to the bottom of the culture dishes. The resulting
suspensions of myocytes were plated onto 24-well dishes at a density of
1x106 cells/mL. Thymidine (0.6 mg/mL) was added
during the first 72 hours to prevent proliferation of
nonmyocytes. Using this method, we routinely obtained enriched
cultures containing more than 95% myocytes, as assayed by
immunofluorescence staining with an anti–myosin
heavy chain antibody.38 The macrophage cell line
J774 was cultured as described previously.39
The investigation was performed in accordance with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act, 1986, published by Her Majesty's Stationery Office, London.
Measurement of Nitrite Levels
Cells were incubated in 0.5% FBS containing DMEM, and nitrite
in the culture medium was measured by mixing 0.5 mL of the medium with
an equal volume of Griess reagent (0.1% naphthylethylenediamine
dihydrochloride and 1% sulfanilamide in 5% phosphoric
acid).40 The absorbance at 550 nm was measured, and
nitrite concentration was determined using a curve calibrated from
sodium nitrite standards. After washing, cells were dissolved in 0.2 mL
of 1% SDS and used for protein assay (BCA protein assay kit; Pierce)
with bovine serum as a standard. Nitrite levels were corrected by
protein measurements, and data are shown as nanomoles per milligram
protein.
Assay for iNOS mRNA
iNOS mRNA expression in cardiac myocytes was analyzed by
Northern blotting, as reported previously.41 Total RNA was
extracted from cardiac myocytes plated in 100-mm culture dishes by the
acid guanidinium thiocyanate–phenol-chloroform method, and 30 µg
aliquots were subjected to electrophoresis on 1% agarose gels
containing formaldehyde and transferred onto nylon filters (Hybond
N+, Amersham International Plc). The filters were
hybridized with a random-primed 32P-labeled mouse
macrophage iNOS cDNA probe that was prepared by reverse
transcription–polymerase chain reaction.41 The hybridized
filters were then washed in 150 mmol/L NaCl, 15
mmol/L sodium citrate, and 0.1% SDS at 65°C and exposed to
Kodak XAR-5 film overnight at -70°C with one intensifying
screen.
Assay for iNOS Protein
iNOS protein was analyzed by
immunoblotting with the anti-iNOS antibody as described
previously.42 Briefly, the cells were lysed in a buffer
containing 50 mmol/L Tris/HCl (pH 7.5), 1
mmol/L EDTA, 1 µmol/L leupeptin, 1
µmol/L pepstatin A, 0.1 mmol/L
phenylmethylsulfonyl fluoride, and 1 mmol/L
dithiothreitol and then sonicated. The homogenates were
then centrifuged at 100 000g for 20 minutes, and
the supernatants were subjected to 10% SDS-PAGE using the buffer
system described by Laemmli.43 The separated proteins were
electrophoretically transferred onto nitrocellulose membranes, and the
nitrocellulose blots were incubated with anti-iNOS antibody for 2
hours, followed by peroxidase-labeled donkey anti-rabbit IgG for 1
hour. Horseradish peroxidase–labeled proteins were visualized by
incubation with peroxidase color development reagents containing the
enzyme substrate 3,3'-diaminobenzidine with NiCl2 used as
an enhancer.
Measurement of [Ca2+]i
[Ca2+]i of cardiocytes were
estimated from the fura-2 fluorescence as previously
described.37 The cells were rinsed with PSS containing
140 mmol/L NaCl, 4.6 mmol/L KCl, 1
mmol/L MgCl2, 2 mmol/L
CaCl2, 10 mmol/L glucose, and 10
mmol/L HEPES, pH 7.4. They were then loaded with 5
µmol/L fura-2 acetoxymethyl ester (fura-2/AM) for 60 minutes
at 37°C. After aspiration of the fura-2/AM solution, the glass slides
were rinsed and then placed in a quartz cuvette at 37°C in a
fluorescence spectrometer (model CAF-100, Japan Spectrometer).
The fluorescence was monitored at 500 nm with excitation
wavelengths of 340 and 380 nm in the ratio mode. From the ratio of
fluorescence at 340 and 380 nm, the
[Ca2+]i was determined as described by
Grynkiewicz et al44 using the following expression:
[Ca2+]i
(nmol/L)=Kdx[(R–Rmin)/(Rmax–R)]X
ß, where R is the ratio of fluorescence of the
sample at 340 and 380 nm, and Rmax and
Rmin are determined by treating the cells with
50 µmol/L digitonin and 10 mmol/L
MnCl2, respectively. The term ß is the ratio of
fluorescence of fura-2 at 380 nm in digitonin and
MnCl2. Kd is the dissociation
constant of fura-2 for Ca2+, assumed to be 224 nm at
37°C.
cAMP Measurement
For determination of intracellular cAMP levels, 0.5
mmol/L IBMX, a cyclic nucleotide phosphodiesterase
inhibitor, was added to each well 30 minutes before the
addition of AVP or forskolin to prevent breakdown of accumulated cAMP.
After incubation with AVP or forskolin for 1 hour, cells were
immediately immersed in 0.2 mL of 0.1N HCl to stop the reaction. Cells
were then collected into glass tubes with a rubber policeman, boiled
for 5 minutes, and then centrifuged at 2500g for 15
minutes at room temperature. The supernatants were decanted, and after
0.05 mL of 50 mmol/L sodium acetate was added to each tube,
cells were kept at -70°C until assayed for cAMP contents. The
pellets were dissolved in 0.2 mL of 1% SDS and kept at 4°C until
assayed for protein. Intracellular cAMP contents were measured with a
commercial enzyme immunoassay kit using the manufacturer's high
sensitivity acetylation protocol (Amersham International
Plc). The lower limit of detection was 2 fmol per well. The values were
normalized to protein content of each well.
Measurement of Protein Kinase C Activity
Cells grown in 24-well dishes were incubated with DMEM
supplemented with 0.5% FBS for 24 hours. After washing twice with PSS,
cells were exposed to AVP for 10 minutes at 37°C. The reaction was
stopped by the addition of 100 µL of extraction solution (20
mmol/L Tris/HCl, 0.5 mmol/L EDTA, 0.5
mmol/L EGTA, 0.5% Triton X-100, 25 µg/mL aprotinin,
and leupeptin, pH 7.5). Cell extracts were centrifuged at
1500g for 5 minutes. The supernatant was then incubated with
25 µmol/L of a synthetic peptide [4-14 amino acids of
bovine myelin basic protein (MBP4-14)] (Sigma Chemical
Co)45 and reaction mixture containing 20
mmol/L Tris/HCl (pH 7.5), 5 mmol/L magnesium
acetate, 0.1 mmol/L CaCl2, 0.5 µg
phosphatidyl serine, 50 ng diolein, and 50 µmol/L
[
-32P]ATP (specific activity; 10 Ci/mmol, New England
Nuclear) for 10 minutes at 30°C. The reaction products were
placed on P-81 paper (Whatman International Ltd) and washed three times
with 20 mL of ice-cold 10% phosphoric acid. The radioactivity was
counted by a liquid scintillation counter (Aloka LSC-671). Specific
radioactivity was obtained by subtracting the radioactivity of the
synthetic peptide-free reaction from the synthetic peptide–directed
radioactivity. Protein kinase C activity was represented as
picomoles of ATP incorporated per milligram protein of cell extracts
for 1 minute.
Statistical Analysis
Data are expressed as mean±SEM of four samples, which
represented at least three separate experiments.
Differences were analyzed by one-way ANOVA combined with
Scheffé's test, and values of P<.05 were considered
to be statistically significant.
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Results |
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Effects of AVP on Nitrite Production
We first investigated the effects of IL-1ß on NO production by cardiac myocytes. Accumulation of nitrite in the medium represents the summation of NOS activity during the time period studied, since NO secreted by cells is rapidly decomposed to the more stable products nitrite and nitrate. As shown in Fig 1A
, treatment of cardiac myocytes with
IL-1ß (10 ng/mL) caused marked accumulation of nitrite in the
culture medium in a time-dependent manner. This IL-1ß–induced
nitrite accumulation was significantly augmented by
simultaneous treatment of the cells with AVP. Fig 1B shows
the dose-response effect of AVP on nitrite production.
Incubation of the cultures with AVP for 24 hours increased nitrite
production by IL-1ß–stimulated cardiac myocytes in a
dose-dependent manner (0.1 to 1000 nmol/L). AVP by itself did
not affect the basal levels of nitrite production.
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), IL-1ß+AVP (1 µmol/L) (
), or vehicle (
)
for 36 hours. B, Dose-dependent effects of AVP on nitrite
production by cardiac myocytes. Myocytes were exposed to 0.1 to
1000 nmol/L AVP in the presence (closed columns) or absence (hatched
columns) of IL-1ß (10 ng/mL) for 24 hours. Nitrite accumulation in
the culture medium was measured, and the values were normalized to the
protein content per dish. Data are mean±SEM of four samples.
*P<.05, 
P<.01 compared with control cells
exposed to IL-1ß alone.
As shown in Fig 2, incubation with the
V1a receptor agonist [Phe2,Ile3,
Orn8]vasopressin for 24 hours also increased nitrite
production by IL-1ß–stimulated cells in a dose-dependent
manner (0.1 to 1000 nmol/L). On the other hand, neither the
V1b receptor agonist
[deamino1,D-3-(pyridyl)Ala2,Arg8]vasopressin
nor the V2 receptor agonist 1-deamino-8-D-AVP
affected nitrite production by IL-1ß–stimulated cells. The
AVP agonists by themselves did not affect the basal levels of nitrite
production (data not shown).
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On the other hand, the V1a receptor antagonist
[d(CH2)51,O-Me-Tyr2,Arg8]vasopressin
dose-dependently abolished the stimulatory effect of AVP on
IL-1ß–induced nitrite production by cardiac myocytes (Fig 3). When 1 µmol/L AVP was
applied, the concentrations of
[d(CH2)51,O-Me-Tyr2,Arg8]vasopressin
required for half-maximal and maximal blockade were approximately 10
nmol/L and 1 µmol/L, respectively.
[d(CH2)51,O-Me-Tyr2,Arg8]vasopressin
did not affect nitrite accumulation induced by IL-1ß alone.
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As shown in Fig 4, the addition of AVP
(1 µmol/L) either 3 or 6 hours after treatment of the
cells with IL-1ß (10 ng/mL) still augmented nitrite
production, although the effect was significantly decreased.
However, no stimulatory effect was observed when AVP was added 12 hours
after IL-1ß treatment.
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Simultaneous incubation with IL-1ß in the presence of the NOS inhibitor L-NMMA (1 mmol/L) or the RNA synthesis inhibitor actinomycin D (5 µg/mL) for 24 hours completely inhibited AVP-induced as well as IL-1ß–induced nitrite production (data not shown).
Effects of AVP on iNOS mRNA and Protein Accumulations
Since the chronological analysis described above strongly
suggested that AVP augmented IL-1ß–induced NO production at
the level of iNOS expression, we examined whether AVP actually induced
increases in iNOS mRNA levels in cardiac myocytes. As shown in Fig 5, unstimulated and AVP-treated myocytes
expressed no detectable iNOS mRNA, whereas exposure to IL-1ß (10
ng/mL) for 24 hours clearly induced its accumulation.
Coincubation with AVP (1 µmol/L) for 24 hours further
augmented the IL-1ß–induced increase in iNOS mRNA accumulation.
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Furthermore, the expression of iNOS protein was analyzed
by immunoblotting with the anti-iNOS antibody. No
immunoreactive band of iNOS was detected in unstimulated cardiac
myocytes (Fig 6). The iNOS protein band
with a molecular mass of 125 kD was clearly apparent after exposure to
IL-1ß for 24 hours, and its accumulation was further increased in the
presence of AVP.
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Effects of AVP on [Ca2+]i and
cAMP
We next investigated the mechanism of the stimulatory effect of
AVP on NO production. The V1a receptor mediates the
mobilization of intracellular Ca2+ and activation of
protein kinase C, whereas the V2 receptor mediates the
formation of cAMP. As shown in Fig 7, AVP
(1 µmol/L) rapidly increased
[Ca2+]i of cardiac myocytes, whereas
preincubation of the cells with the V1a receptor
antagonist
[d(CH2)51,O-Me-Tyr2,Arg8]vasopressin
(1 µmol/L) for 5 minutes completely blocked the
AVP-induced increase in [Ca2+]i.
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The Table summarizes the effects of AVP,
V1a, and V1b agonists and V1a
antagonist on [Ca2+]i of cardiac
myocytes. Both AVP and the selective V1a receptor agonist
[Phe2,Ile3,Orn8]vasopressin
significantly increased [Ca2+]i, and the
effect of AVP was completely abolished in the presence of the
V1a receptor antagonist
[d(CH2)51,O-Me-Tyr2,Arg8]vasopressin.
There was no significant increase in [Ca2+]i
by the V1b receptor agonist
[deamino1,D-3-(pyridyl)Ala2,Arg8]vasopressin.
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On the other hand, the addition of AVP (1 µmol/L) to the
culture did not affect cytosolic cAMP contents of cardiac myocytes,
although forskolin (1 µmol/L) significantly increased
cAMP contents (Fig 8), suggesting that
the functional V2 receptor is not located on rat cardiac
myocytes.
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Involvement of Protein Kinase C
In vascular smooth muscle cells, the V1a receptor
activation is coupled to phospholipase C–mediated
phosphoinositide hydrolysis and activation of protein
kinase C.18 It has been shown that activation of protein
kinase C causes an upregulation of iNOS expression in cardiac
myocytes,46 47 and thus it is possible that this signaling
pathway is involved in the stimulatory effect of AVP on NO
production by cardiac myocytes. We examined the effect of AVP
on NO synthesis in control and protein kinase C–depleted cardiac
myocytes. It has been shown that protein kinase C in cardiac myocytes
is downregulated by pretreatment with PMA.48 Thus, cells
were exposed to PMA (1 µmol/L) for 24 hours in 10% FBS
containing DMEM and then incubated in 0.5% FBS containing DMEM with
AVP (1 µmol/L), IL-1ß (10 ng/mL), or PMA (100
nmol/L) for a further 24 hours. As shown in Fig 9, in control cells not preincubated with
PMA, nitrite levels were significantly increased 24 hours after the
addition of IL-1ß. The addition of AVP or PMA further augmented
nitrite accumulation in IL-1ß–stimulated cells. On the other hand,
in cells preincubated with PMA for 24 hours, IL-1ß still increased
nitrite levels, but the addition of fresh PMA caused no change in
nitrite levels, which is consistent with the functional
depletion of protein kinase C activity. The IL-1ß–induced nitrite
levels were not significantly affected by AVP in protein kinase
C–depleted cells.
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We further tested the effects of the protein kinase C
inhibitor calphostin C on the effect of AVP. As shown in
Fig 10, calphostin C dose-dependently
abolished the stimulatory effect of AVP on IL-1ß–induced nitrite
production by cardiac myocytes.
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We then measured protein kinase C activity in cardiac myocytes. As
shown in Fig 11, the addition of AVP
(1 µmol/L) increased protein kinase C activity in cardiac
myoctes, and its effect was significantly inhibited in the presence of
calphostin C.
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Discussion |
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The present study was designed to examine whether AVP modulated NO production by cardiac myocytes. Although AVP alone had no effect on nitrite production, it markedly augmented IL-1ß–induced nitrite production in time- and concentration-dependent manners. The stimulatory effect of AVP was reduced when AVP was added several hours after the addition of the cytokine and in the presence of L-NMMA or actinomycin D, suggesting that the upregulation of nitrite production by AVP is due to enhanced induction of iNOS. Indeed, AVP further augmented IL-1ß–induced increases in iNOS mRNA and protein levels.
In addition to its potent antidiuretic action, AVP exerts both vasoconstricting and vasodilating effects via V1a and V2 receptors on the vascular tissue, respectively.49 50 Recently, with the reverse transcription-polymerase chain reaction analysis, Hirasawa et al51 reported that V1a receptor mRNA is detected not only in rat brain, liver, and kidney but also in heart. Also in humans, very recently, it has been reported that V1a receptor mRNA is expressed in the heart.52 However, no biological function has been demonstrated in the heart in association with agonists coupling to the V1a receptor. In the present study, AVP augmented nitrite production by IL-1ß–stimulated cardiac myocytes, and its effect was completely abolished by the V1a receptor antagonist [d(CH2)51,O-Me-Tyr2,Arg8]vasopressin, whereas neither the V1b receptor agonist [deamino1,D-3-(pyridyl)Ala2,Arg8]vasopressin nor the V2 receptor agonist 1-deamino-8-D-AVP affected NO synthesis in IL-1ß–stimulated cells. In addition, by measuring [Ca2+]i and cAMP levels of cardiac myocytes, we revealed that rat cardiac myocytes possess functional V1a receptors but not V1b or V2 receptors.
We have previously observed that the protein kinase C pathway is involved in the induction of iNOS in cytokine-stimulated cardiac myocytes.46 Since V1a receptor activation is coupled to phospholipase C–mediated phosphoinositide hydrolysis,26 it is possible that this signaling pathway is involved in the stimulatory effect of AVP on cytokine-induced iNOS expression in cardiac myocytes. Therefore, we examined the potential role of protein kinase C in AVP-induced enhancement of IL-1ß–induced NO production with the aid of the protein kinase C–activating phorbol ester PMA. Recently, Clerk et al48 demonstrated that prolonged exposure of myocytes to 1 µmol/L PMA caused essentially complete downregulation of protein kinase C. In this study, when protein kinase C was downregulated by pretreatment with 1 µmol/L PMA for 24 hours, the effect of AVP, as well as that of PMA, was completely abolished. The effect of AVP on NO production was also significantly inhibited by the protein kinase C inhibitor calphostin C. Furthermore, the addition of AVP increased protein kinase C activity in cardiac myocytes, and its effect was significantly inhibited in the presence of calphostin C. These results indicate that activation of protein kinase C mediates, at least partially, the effect of AVP on IL-1ß–induced NO production.
The findings presented here do not address the molecular mechanism by which AVP altered the iNOS mRNA levels in IL-1ß–stimulated cardiac myocytes. Changes in either transcription or in mRNA stability may account for the changes in mRNA levels. From the potent inhibitory action of actinomycin D and the lag period of several hours before the onset of iNOS activity, transcriptional activation of iNOS expression seems to be a very likely explanation for the observations described here. However, nuclear run-on experiments will be necessary to directly assess rates of transcription of the iNOS gene.
In cardiac myocytes, eNOS, as well as iNOS, is
expressed.53 Balligand et al53 have shown
that eNOS mRNA is constitutively expressed in unstimulated rat cardiac
myocytes and that its abundance is markedly decreased after 24 hours of
treatment with cytokines. On the other hand, in our experiments
using the iNOS cDNA and antibody, no detectable signal was observed in
unstimulated myocytes, and signals became clearly detectable after 24
hours of treatment with IL-1ß and AVP (Figs 5
and 6). Therefore, we
assume that IL-1ß and AVP increase NO production by cardiac
myocytes via iNOS induction; however, it is still uncertain whether AVP
also affects eNOS activity in cardiac myocytes. Another limitation of
the present study is that we measured only nitrite as a stable
metabolite of NO, although NO released by the myocytes is metabolized
to both nitrite and nitrate.
In view of the findings of the present study, it is likely that AVP-augmented IL-1ß–induced NO synthesis in the heart could contribute to the intrinsic cardiac depression or injury that often characterizes immune- and inflammation-mediated myocardial diseases.14 Previous studies demonstrated that plasma AVP levels were elevated in patients with congestive heart failure.33 34 35 Benedict et al35 reported a significant negative correlation between ejection fraction of the left ventricle and increased plasma AVP levels in patients with heart failure. Circulating AVP levels (1.6 to 2.7 pmol/L) in patients with congestive heart failure might be lower than those used in the present study35 ; however, recently, it has been shown that, in addition to the well-understood endocrine system, there is an independent peripheral AVP system in several organs including the vascular tissue.54 55 These observations are consistent with, although do not prove, the hypothesis that locally as well as systemically produced AVP may act as an endogenous enhancer of cytokine-induced NO production by the heart in certain immunological and inflammatory conditions, including postcardiac transplantation, cardiomyopathy, myocarditis, ischemia-reperfusion injury, and congestive heart failure.
In conclusion, the present study revealed that the heart may be a target organ for AVP, and AVP modulates NO synthesis in the heart under cytokine-stimulated conditions. However, further studies are required to determine whether the effects of AVP on NO production described here contribute to the development of cardiac dysfunction or injury in the above pathological states in vivo.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by a grant from the Ministry of Education, Culture, and Science of Japan (#8670821).
Received November 7, 1996; first decision December 13, 1996; accepted April 15, 1997.
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